Post-translational histone modifications are essential to the proper regulation of nearly all DNA-mediated processes, whereas improper modifications lead to numerous disease states. Histone modifications exist in the context of chromatin, a complex assembly of DMA, histones (H2A, H2B, H3, and H4), and chromatin- associated proteins. Chromatin exhibits a complex hierarchy of higher-order structures, and our long-term goal is to elucidate the interplay between higher-order chromatin structure and histone modifications. This proposal details studies that both capitalize on our ability to generate chromatin model systems containing uniformly modified H3 and H4 histones and on our recent discovery that histone H4 lysine 16 acetylation is sufficient to directly disrupt both intra- and intermolecular chromatin compaction. The purpose of this work is to gain an in depth understanding of how H3 and H4 histone tail acetylation, H2B carboxy- terminal triacetylation, and H2B monoubiquitination affect higher-order chromatin structure individually and in combination, as well as how these marks influence subsequent histone modifications. Three specific aims have been proposed to address these questions. First, we will utilize various chromatin model systems containing varying patterns and levels of histone H3 and H4 acetylation to understand the mechanistic details of how histone tail acetylation affects higher-order chromatin structure. Second, we will elucidate how changes in higher-order chromatin structure direct histone tail acetylation using a combination of biophysical, enzymatic and genetic approaches. Third, we will develop novel techniques for introducing H2B carboxy- terminal modifications to determine their structural and functional roles. Completion of these aims will help to define how post-translational histone modifications and higher-order chromatin structure directly influence one another. By extending our understanding of the role of histone modifications into an area that has not previously been intensively studied, we will obtain a better mechanistic understanding of the normal role of these modifications. Additionally, this understanding can lead to a better appreciation of how improper utilization of these modifications results in diseases, including various cancers and congenital defects, leading to better disease diagnosis and treatment.